Single molecule detection systems and methods

• US 20030186255A1
• Filed: 06/05/2002
• Published: 10/02/2003
• Est. Priority Date: 06/06/2001
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Li & S Company Limited (Yesco Holdings Co., Ltd.)
• Inventors: Bashford, Gregory R., Williams, John G.K.
• Granted Patent: US 7,118,907 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/301 Pyrophosphate (PPi)
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

A microfluidic system is provided that includes a substrate, a first microchannel disposed in the substrate for providing a reactant to a reaction zone, a second microchannel disposed in the substrate, and a third microchannel disposed in the substrate, the third microchannel providing fluid communication between the first and second microchannels. The system also typically includes first and second electrodes, positioned at opposite ends of the second microchannel, for providing an electric field within the second microchannel. In operation, when the reactant is in the reaction zone, a reaction product is produced having a net electric charge different from the electric charge of the reactant.

Claims

1. A microfluidic system, comprising:

a substrate;

a first microchannel disposed in the substrate for providing a reactant to a reaction zone;

a second microchannel disposed in the substrate, wherein at least a portion of the second microchannel is in a detection region;

a third microchannel disposed in the substrate, the third microchannel providing fluid communication between the first and second microchannels, wherein at least a portion of the third microchannel defines a portion of the reaction zone; and

first and second electrodes, positioned at opposite ends of the second microchannel, for providing an electric field within the second microchannel,

wherein when the reactant is in the reaction zone a reaction product is produced having a net electric charge different from the electric charge of the reactant, wherein the reaction product is separated from the reactant, and wherein the electric field in the second microchannel causes the reaction product to flow into the detection region.

2. The system of claim 1, wherein the reactant is provided on a solid phase.

3. The system of claim 2, wherein the solid phase is one of a microbead and a microparticle.

4. The system of claim 2, wherein the reactant includes a nucleic acid complex immobilized on the solid phase in a single molecule configuration.

5. The system of claim 2, wherein the solid phase is magnetic or magnetizable, the device further including a magnetic field generating element positioned proximal the reaction zone of the substrate for holding the solid phase.

6. The system of claim 5, wherein the magnetic field generating element includes one of a permanent magnet and one or more electric coils.

7. The system of claim 1, wherein the net electric charge of the reaction product has an opposite polarity to the electric charge of the reactant.

8. The system of claim 1, wherein the net electric charge of the reaction product has the same polarity as the electric charge of the reactant, but has a charge magnitude different that the charge magnitude of the reactant.

9. The system of claim 8, wherein the reactant product is separated from the reactant due to different mobilities in a bulk fluid flow away from the detection region in the second microchannel based on the difference in charge magnitude between the reactant and the reaction product.

10. The system of claim 1, wherein the reactant includes a target nucleic acid.

11. The system of claim 1, wherein the reactant includes a charge-switch nucleotide probe.

12. The system of claim 11, wherein the nucleotide probe includes a terminal phosphate with a fluorophore attached thereto.

13. The system of claim 12, wherein the probe is a nucleotide triphosphate and the terminal phosphate includes a fluorophore attached thereto.

14. The system of claim 11, wherein the reaction is one of a molecular cleavage reaction and an enzymatic cleavage reaction.

15. The system of claim 1, further including a pressure inducing mechanism for inducing fluid flow in the first microchannel to facilitate delivery of the reactant to the reaction zone.

16. The system of claim 15, further including a fourth microchannel in fluid communication with the first microchannel, and wherein the pressure generating mechanism induces a pressure gradient at the intersection of the first and fourth microchannels to facilitate delivery of the reactant from the fourth microchannel to the first microchannel.

17. The system of claim 16, further including an input port coupled to one end of the fourth microchannel for providing the reactant to the fourth microchannel.

18. The system of claim 17, wherein the reactant has a net electric charge, the device further including third and fourth electrodes positioned at opposite ends of the fourth microchannel relative to the intersection with the first microchannel, for providing an electric field within the fourth microchannel.

19. The system of claim 17, further including a detector proximal the detection region and a feedback circuit coupled to the detector.

20. The system of claim 19, wherein the feedback circuit is coupled to a feed source at the input port, and wherein the feedback circuit causes the feed mechanism to adjust the rate at which reactant is provided to the fourth microchannel in response to a detector signal.

21. The system of claim 19, wherein the feedback circuit is coupled to the pressure generating mechanism, and wherein the feedback circuit causes the pressure generating mechanism to adjust the fluid flow rate in the first microchannel in response to a detector signal.

22. The system of claim 1, further including a detector position proximal the detection region.

23. The system of claim 21, wherein the detector includes one of a fluorescence detector and a total internal reflection fluorescence microscopy (TIRFM) detector.

24. The system of claim 1, wherein the system is configured such that there is substantially no electric field within the third microchannel.

25. The system of claim 1, wherein the system is configured such that there is negligible electric field within the third microchannel.

26. The system of claim 1, wherein the second microchannel has a depth within at least the detection region of between about 0.5 μ

m and about 1.0 μ

m.

27. The system of claim 1, wherein the substrate is one of a PDMS substrate, a fused-silica substrate and a glass substrate.

28. The system of claim 1, wherein any detectable reactant entering the second microchannel is biased away from the detection region by the electric field within the second microchannel.

29. The system of claim 1, further including means for generating an evanescent excitation field within the detection region of the second microchannel, and wherein the second microchannel has a depth within at least the detection region that is less than approximately four times the exponential decay constant of the evanescent excitation field within the detection region.

30. A microfluidic device useful for analyzing a reaction, the device comprising:

a substrate;

at least two microchannels disposed on the substrate, wherein said microchannels are in fluid communication with each other;

an immobilization zone within a first one of said microchannels, wherein at least a portion of a reactant is immobilized proximal the immobilization zone to allow for a reaction to occur so as to produce a reaction product; and

a detection zone within a second one of said microchannels,

wherein there is substantially no electric field within the first microchannel proximal the immobilization zone, and wherein a biasing force moves the reaction product into the detection zone within the second microchannel.

31. The device of claim 30, wherein the biasing force includes one of an electric field, a pressure field and a combination of an electric field and a pressure field.

32. The device of claim 30, wherein the biasing force includes a pressure field in the first microchannel and an electric field within the second microchannel.

33. The device of claim 30, wherein the reaction occurs in a reaction zone proximal the immobilization zone.

34. The device of claim 33, wherein at least a portion of the reaction zone is within the first microchannel.

35. The device of claim 33, wherein at least a portion of the reaction zone is within the second microchannel.

36. The device of claim 33, further including a third microchannel fluidly coupling the first and second microchannels, wherein at least a portion of the reaction zone is within the third microchannel.

37. The device of claim 36, wherein there is substantially no electric field within said third microchannel.

38. The device of claim 33, wherein at least a portion of the reaction zone is in a flow path between the immobilization zone and the detection zone.

39. The device of claim 30, wherein the reactant is provided on a solid phase.

40. The device of claim 39, wherein the reactant includes a nucleic acid complex immobilized on the solid phase in a single molecule configuration.

41. The device of claim 39, wherein the solid phase is magnetic or magnetizable, the device further including a magnetic field generating element positioned proximal the immobilization zone for holding the solid phase.

42. The device of claim 41, wherein the magnetic field generating element includes one of a permanent magnet and one or more electric coils.

43. The device of claim 30, wherein the reaction product has a net charge different from the net charge of the reactant.

44. The device of claim 43, wherein the net electric charge of the reaction product has an opposite polarity to the electric charge of the reactant.

45. The device of claim 43, wherein the net electric charge of the reaction product has the same polarity as the electric charge of the reactant, but has a charge magnitude different that the charge magnitude of the reactant.

46. The device of claim 45, wherein the reactant product is separated from the reactant due to different mobilities in a bulk fluid flow away from the detection zone in the second microchannel based on the difference in charge magnitude between the reactant and the reaction product.

47. The device of claim 30, wherein the reactant includes a charge-switch nucleotide probe.

48. The device of claim 47, wherein the nucleotide probe includes a terminal phosphate with a fluorophore attached thereto.

49. The device of claim 47, wherein the probe is a nucleotide triphosphate and the terminal phosphate includes a fluorophore attached thereto.

50. The system of claim 30, wherein the reaction is one of a molecular cleavage reaction and an enzymatic cleavage reaction.

51. The device of claim 30, wherein the second microchannel has a depth within at least the detection zone of between about 0.5 μ

m and about 1.0 μ

m.

52. The device of claim 30, further including means for generating an evanescent excitation field within the detection zone, and wherein the second microchannel has a depth within at least the detection zone that is less than approximately four times the exponential decay constant of the evanescent excitation field within the detection zone.

53. The device of claim 30, further comprising a third microchannel, coupled to the second microchannel between the immobilization zone and the detection zone, for supplying a second reactant material to the reaction zone.

54. The device of claim 30, wherein the reaction zone is sufficiently close to the detection zone so as to maintain the order of reaction product produced in the reaction as it enters the detection zone.

55. The device of claim 30, wherein the reaction zone is not proximal the detection zone, and wherein the biasing force is sufficiently strong so as to maintain the order of reaction product produced in the reaction as it enters the detection zone.

56. A method of sequencing or genotyping a nucleic acid in a microfluidics device having at least two microchannels disposed on a substrate in fluid communication with each other, said method comprising:

immobilizing a nucleic acid complex proximal an immobilization region within a first one of said microchannels;

providing a polymerase and plurality of NP probes to a reaction region proximal the immobilization region under conditions permitting target dependent polymerization of said plurality of NP probes, thereby providing a product; and

separating the product from polymerization reactants into a detection region in a second one of the microchannels using a biasing force sufficient to interact with the product; and

detecting the product.

57. The method of claim 56, wherein the nucleic acid complex is immobilized on a solid phase in a single molecule configuration, and wherein immobilizing includes immobilizing the solid phase proximal the immobilization region such that the nucleic acid complex is proximal the reaction region.

58. The method of claim 57, wherein the solid phase is magnetic or magnetizable, and wherein immobilizing the solid phase includes generating a magnetic field proximal the immobilization region so as to hold the solid phase in place.

59. The method of claim 56, wherein the product includes fluorophores, and wherein detecting includes exciting the fluorophores and detecting the fluorescent signals emitted thereby.

60. The method of claim 59, wherein exciting includes generating an evanescent excitation field in the detection region.

61. The method of claim 60, wherein the depth of the second microchannel within the detection region is less than approximately three times the exponential decay constant of the evanescent excitation field within the detection region.

62. The method of claim 56, wherein the product has a different net charge than the NP probe, and wherein separating includes generating an electric field within the second microchannel.

63. The method of claim 62, wherein the polarity of charge of the NP probes and the product is the same but the magnitude of the charge of the product is different, whereby the product is separated due to a greater mobility in bulk fluid flow in the second microchannel due to the difference in charge magnitude.

64. A microfluidic device useful for analyzing a reaction, the device comprising:

a substrate;

a first microchannel disposed on the substrate,

a plurality of second microchannels disposed on the substrate, each in fluid communication with the first microchannel;

a plurality of immobilization zones within the first microchannel, each immobilization zone corresponding to one of said plurality of second microchannels, wherein a reactant is immobilized proximal each immobilization zone to allow for a reaction proximal each immobilization zone to produce a reaction product; and

a detection zone within each of the plurality of second microchannels,

wherein there is substantially no electric field within the first microchannel proximal each immobilization zone, and wherein a biasing force moves the corresponding reaction product into the detection zone within each second microchannel.

65. The device of claim 64, wherein each reaction occurs within a reaction zone within each corresponding second microchannel.

66. The device of claim 65, further comprising a third microchannel, coupled to each second microchannel proximal each reaction zone, for providing a second reactant to each reaction zone.

67. The device of claim 64, further including a plurality of third microchannels, each coupling the first microchannel to a corresponding second microchannel.

68. The device of claim 67, wherein each reaction occurs within a corresponding reaction zone, and wherein each reaction zone is at least partially located within the corresponding third microchannel.

69. The device of claim 68, wherein there is substantially no electric field within each third microchannel.

70. The device of claim 64, wherein the biasing force includes one of an electric field, a pressure field and a combination of an electric field and a pressure field.

71. The device of claim 64, wherein the biasing force includes a pressure field in the first microchannel and an electric field within each second microchannel.

72. The device of claim 64, wherein each reactant is provided on a solid phase.

73. The device of claim 72, wherein each reactant includes a nucleic acid complex immobilized on the solid phase in a single molecule configuration.

74. The device of claim 72, wherein the solid phase is magnetic or magnetizable, the device further including a magnetic field generating element positioned proximal each immobilization zone for holding the solid phase.

75. The device of claim 74, wherein each magnetic field generating element includes one of a permanent magnet, and one or more electric coils.

76. The device of claim 64, wherein each reaction product has a net charge different from the net charge of the corresponding reactant.

77. The device of claim 76, wherein the net electric charge of each reaction product has an opposite polarity to the electric charge of the corresponding reactant.

78. The device of claim 76, wherein the net electric charge of each reaction product has the same polarity as the electric charge of the corresponding reactant, but has a charge magnitude different that the charge magnitude of the corresponding reactant.

79. The device of claim 78, wherein each reactant product is separated from the corresponding reactant due to different mobilities in a bulk fluid flow away from the detection zone in the corresponding second microchannel based on the difference in charge magnitude between the reactant and the reaction product.

80. The device of claim 64, wherein each reactant includes a charge-switch nucleotide probe.

81. The device of claim 80, wherein each nucleotide probe includes a terminal phosphate with a fluorophore attached thereto.

82. The device of claim 80, wherein each probe is a nucleotide triphosphate and the terminal phosphate includes a fluorophore attached thereto.

83. The device of claim 64, wherein each reaction is one of a molecular cleavage reaction and an enzymatic cleavage reaction.

84. The device of claim 64, wherein each second microchannel has a depth within at least the detection zone of between about 0.5 μ

m and about 1.0 μ

m.

85. The device of claim 64, further including means for generating an evanescent excitation field within each detection zone, and wherein each second microchannel has a depth within at least the detection zone that is less than approximately four times the exponential decay constant of the evanescent excitation field within the detection zone.

86. The device of claim 64, wherein each reaction zone is sufficiently close to the corresponding detection zone so as to maintain the order of reaction product produced in the reaction as it enters the detection zone.

87. The device of claim 64, wherein each reaction zone is not proximal the corresponding detection zone, and wherein the biasing force is sufficiently strong so as to maintain the order of reaction product produced in the reaction as it enters the detection zone.

88. The device of claim 64, wherein the first microchannel has a substantially circular configuration with each of said second microchannels being oriented substantially radially inward relative to the first microchannel.

89. The device of claim 64, further including a plurality of third microchannels, each coupling the first microchannel to a corresponding second microchannel, wherein each third microchannel is oriented substantially radially inward relative to the first microchannel.

90. The device of claim 88, wherein each detection zone within each of the plurality of second microchannels is located proximal the center point of the circular configuration, and wherein an illumination source illuminates all detection zones simultaneously.